BACKGROUND: Assessment of heart rate variability (HRV) is an effective non-invasive tool to obtain data on cardiac autonomic modulation and may be assessed by a range of devices, including mobile applications. Objective: This study aimed to validate a smartphone application by comparing the R-R intervals (RRi) obtained by the app with a classic electrocardiogram (ECG)-derived reference condition Methods: Fifteen asymptomatic adults (24.9+/-3.4 years) underwent an orthostatic challenge during which RRi were simultaneously recorded by a freeware smartphone application and by an ECG recorder. Pearson correlation coefficients (r) and coefficients of determination (r (2)) were calculated to determine the degree of association between the two electronic devices. Two-way repeated measures analysis of variance and Bland-Altman analysis were used to calculate the measurement consistency and agreement, respectively, between the two methods. Effect size was also used to estimate the magnitude of the differences. RESULTS: The number of RRi from asymptomatic adults recorded by the ECG and by the free smartphone application was similar at rest in supine position (13,149 vs. 13,157; P = 0.432) and during orthostatic challenge (10,666 vs. 10,664 P = 0.532). RRi in milliseconds from both devices presented a near perfect correlation in the supine position (r = 0.999; Confidence Interval [CI] at 95%: 0.999-0.999; P < 0.0001) and during orthostatic challenge (r = 0.988; 95% CI: 0.988-0.989; P < 0.0001). A negative bias of -0.526 milliseconds (95% limits of agreement [LoA] from -4.319 to 3.266 milliseconds) was observed in supine position between ECG and the smartphone application. On the other hand, a positive bias of 0.077 milliseconds (95% LoA from -10.090 to 10.240 milliseconds) during the orthostatic challenge was observed. CONCLUSIONS: Our results cross-validated a freeware smartphone application with the ECG-derived reference condition for asymptomatic adults at rest in the supine position and during orthostatic challenge.